We have developed a model for studying tolerance to persistent low dose antigen in vivo, which results in the generation of a large number of anergic (hyporesponsive) T cells. We call this state adaptive tolerance. We inject CD4+, cytochrome c-specific T cells from a T cell receptor transgenic mouse on a Rag2-/- background (a monospecific T cell population) into a second transgenic mouse (called mPCC) expressing the cytochrome c antigen under the control of the MHC class I promoter and an immunoglobin heavy chain enhancer. Within 24 hours after transfer, the T cells are all activated by the antigen (as evidenced by an increase in size and expression of CD69), and proliferate extensively for several days, increasing in number about 100-fold. This expansion is followed by a deletional phase during which 50% of the cells disappear. Finally, the population reaches a steady state level in which the cells appear to be refractory to restimulation in vivo and in vitro. In this adaptive tolerant state, cytokine responses to high doses of antigen in vitro are inhibited 90%. However, in vivo BrdU labeling shows a slow T cell turnover of about 5% per day and B cell help is still sufficient for the mice to eventually develop a mild form of chronic autoimmune arthritis. The hyporesponsive state is reversible if the cells are transferred again into a second host not expressing the antigen. If the retransfer is into a host expressing the antigen, the T cells remain hyporesponsive and slowly decrease their IL-2 and IFN gamma production by another 6-10 fold over 3-4 weeks. This deeper state of anergy suggests that the tolerance process is adaptable to different levels.[unreadable] During the past year the lab has focused on the role of co-stimulation in adaptive tolerance. The induction of unresponsiveness in vivo was initially thought to stem from antigen presentation in the absence of co-stimulation since most dendritic cells in unmanipulated animals are in a resting state and dont express high levels of co-stimulatory molecules. However, the initial response of the nave T cells following transfer was a massive expansion and differentiation toward a Th-1 effector phenotype. To determine whether this might be mediated by more efficient co-stimulation available in vivo, we first crossed the cytochrome TCR transgenic mouse onto a CD28 knock-out background. Surprisingly there was little impairment of the expansion phase or the early differentiation to IFN gamma production. IL-2 production was initially 10 fold lower in KO cells as reported by others; but this level was decreased another 10 fold following what appeared to be the normal induction of anergy. Unlike the wild type cells, however, the survival of the KO cells was impaired. This was associated with a decreased turnover of the cells as measured by BrdU-labeling and impairment in their proliferative response on re-transfer into a second antigen-bearing host. If the initial cell transfer was done into a T cell replete (intact) host, the knock out cells showed no differences from wild type cells in either expansion or survival. These observations suggest that CD28 co-stimulation is not required for either the initial expansion phase or the induction of anergy in vivo. [unreadable] To determine whether augmentation of costimulation could prevent the induction of adaptive tolerance, we injected ligands that stimulate various toll receptors after the T cells were transferred into the antigen-bearing host. The addition of lipopolysaccharide, poly-IC and/or CpG in this context, acutely or chronically, failed to significantly alter the tolerogenic phenotype in the responding T cells. This contrasted with the ability of such adjuvants to improve T cell responses to soluble peptide immunizations. We reconciled this difference by uncovering a poorly appreciated property of TLR ligands, which extends the duration of soluble antigen presentation in vivo by an additional two to three days. Finally, we were able to replace the requirement for TLR-mediated APC activation in soluble-antigen-induced T cell expansion and differentiation, by maintaining the antigen depot in vivo using repeated immunizations. These data suggest a novel process by which TLR ligands modulate T cell responses to acute antigens, without disrupting the induction of tolerance to persistent self antigens.